Circulating autoantibodies to GAD65, the Mr 65,000 isoform of glutamic acid decarboxylase, are one of the most important markers of the autoimmune process that results in type I diabetes mellitus. A growing body of evidence implicates GAD65, and, in mice, GAD67, as key initiating autoantigens in the autoimmune process. Previously it was thought that GAD65 was the only GAD isoform expressed in human islets. We have shown, however, that human islets also produce a GAD67 variant, GAD25, as the result of an alternative splicing event. Preliminary data suggest that certain young children with newly diagnosed diabetes produce autoantibodies to GAD25. Other data suggest that release of GAD65 from beta cells, perhaps along with GAD25, may help trigger islet autoimmunity. Our central hypothesis is that GAD65 functions as a major autoantigen in type 1 diabetes because its vesicular association makes it susceptible to stress-induced release and also that GAD25 may function as an autoantigen because it, too, can be released. The specific aims of this proposal are 1) to test the hypothesis that GAD65 and GAD25 are uniquely released from human islet beta cells by physiologic stressors; 2) to test the hypothesis that membrane-associated GAD is the intracellular source of released GAD molecules and begin to characterize the mechanism of release and 3) to test the hypothesis that GAD25, by virtue of its localization and regulation, is a potential autoantigen in type 1 diabetes. The long-term objective of this research is to better understand the mechanisms underlying the early immune reaction to GAD65 and the role played by GAD25. Knowledge of the factors that precipitate islet autoimmunity will be vital for the development of therapies to prevent or inhibit beta cell destruction and could result in new treatments to help protect transplanted islets from immune-mediated destruction. This research will prepare the applicant for an academic career as an independent investigator in the field of type I diabetes. He will obtain further training in molecular biology, learn to conduct population-based studies and become acquainted with the key methodologies and approaches used to study islet cell biology and physiology and diabetogenic autoreactivity. The transition to independence will be facilitated by the especially rich training environment afforded, first, by the R.H. Williams Laboratory, headed by Ake Lernmark, where the research will be carried out and, second, by the large surrounding community of highly productive diabetes researchers, all in close proximity, of which the Williams Laboratory is a member.